Effects of Acanthus ebracteatus Vahl. extract and verbascoside on human dermal papilla and murine macrophage

Androgenic alopecia is a common type of hair loss, usually caused by testosterone metabolism generating dihydrotestosterone and hair follicular micro-inflammation. These processes induce dermal papilla cells to undergo apoptosis. Currently approved effective medications for alopecia are Finasteride, an oral 5α-reductase inhibitor, Minoxidil, a topical hair growth promoter, and Diclofenac, an anti-inflammatory agent, all of which, however, have several adverse side effects. In our study, we showed the bioactivity of Acanthus ebracteatus Vahl. (AE) extract performed by 95% ethanol, and verbascoside (VB), a biomarker of AE extract. Both AE extract and VB were studied for their effects on dermal papilla cell viability and the cell cycle by using MTT assay and flow cytometry. The effect of an anti-inflammatory activity of AE extract and VB on IL-1β, NO, and TNF-α, released from LPS induced RAW 264.7 cells, and IL-1α and IL-6 released from irradiated dermal papilla cells were detected using ELISA technique. The preventive effect on dermal papilla cell apoptosis induced by testosterone was determined by MTT assay. In controlled in vitro assays it was found that AE extract and VB at various concentrations induced dermal papilla cell proliferation which was indicated by an increase in the number of cells in the S and G2/M phases of the cell cycle. AE extract at 250 µg/mL concentration or VB at 62.50 µg/mL concentration prevented cell apoptosis induced by testosterone at a statistically significant level. In addition, both AE extract and VB greatly inhibited the release of pro-inflammatory cytokines from RAW 264.7 and dermal papilla cells. The release of IL-1β, TNF-α, and NO from RAW 264.7 cells, as well as IL-1α and IL-6 from dermal papilla cells, was also diminished by AE extract 250 µg/mL and VB 125 µg/mL. Our results indicate that AE extract and VB are promising ingredients for anti-hair loss applications. However, further clinical study is necessary to evaluate the effectiveness of AE extract and VB as treatment for actual hair loss.


Results
Plant extract preparation and VB analyzed using HPLC. AE extracts were prepared using 95% ethanol, 50% ethanol, water and hexane. The amount of VB in each extract was measured using the HPLC technique. The indicative HPLC method linearity was defined as having r 2 greater or equal to 0.9997 using concentrations ranging from 19.53 to 1000.00 µg/mL. The limit of detection (LOD) value of VB was 0.04 pg/mL while the limit of quantification (LOQ) was 0.12 pg/mL. This method was precise with intra-day relative standard deviation (%RSD) less than 5.84% and inter-day values less than 5.05%. The method also showed high accuracy with percentage recovery ranging from 90 to 110%. Therefore, this HPLC method was accurate and reliable for the determination of the amount of VB in AE extract. Sample chromatogram of VB in AE extract with 95% ethanol is presented in Fig. 1.
VB contents in various AE extracts are shown in Table 1. The highest content of VB was found in an extract using 95% ethanol (9.58 ± 0.65%). In contrast, VB could not be detected in the AE extract using hexane. These results suggest that AE extract in 95% ethanol was a promising candidate for further study because it had the highest content of VB.
Determination of 5α-Reductase Inhibition. One hundred micrograms per milliliter of each AE extract was determined for 5α-reductase inhibitory activity. Finasteride was used as the positive control. Their inhibitory activities are shown in Table 2. The comparison of the inhibition activities indicated that AE extract with 95% ethanol showed the highest inhibition (38.26 ± 4.90%), followed by AE extract with hexane (36.92 ± 2.41%), 50% ethanol (13.72 ± 4.88%) and water (6.75 ± 4.20%) while, surprisingly, no inhibitory activity was found for VB. AE extract with 95% ethanol was selected for further bioactivity studies of both VB content and 5α-reductase inhibitory activity. www.nature.com/scientificreports/ Cytotoxic effect of AE extract and VB on human dermal papilla cells. The cytotoxic effect of AE extract with 95% ethanol on dermal papilla cells was evaluated by MTT assay. The results indicated that AE extract and VB at a concentration of 0.98 to 500 µg/mL did not show any cytotoxic effects on dermal papilla cells (Fig. 2). All of the treated cells presented relative cell viability of more than 80% compared to the control cells 30 . Interestingly, VB at 31.25 to 500 µg/mL concentration and AE extract at 62.50 to 500 µg/mL concentration showed significantly increased relative cell viability of dermal papilla cells. This means that AE extract and VB at these concentrations might be able to activate dermal papilla cell proliferation.
Cell cycle analysis. Dermal papilla cells were treated with AE extract of 95% ethanol or VB at a concentration between 31.25 and 500 µg/mL for 24 h. They were then analyzed at different points in the cell cycle with propidium iodide staining. AE extract showed different effects on the dermal papilla depending on concentrations (Fig. 3a). The dermal papilla cell growth (G1 phase) was stimulated by AE extract 500 µg/mL. However, the preparation for cell division (G2/M phase) was lower than that of the control cells. On the contrary, AE extract at 250 µg/mL and 125 µg/mL concentrations showed a significantly increased number of cells in the G2/M   Inhibition effects of AE extract and VB on testosterone-induced cell apoptosis. Cell apoptosis was determined in terms of cell viability. The concentration of 250 µg/mL 95% ethanol AE extract was the optimal value identified in our cell cycle analysis and was therefore selected for these tests. Two hundred micromolar concentrations of testosterone caused a decrease in dermal papilla cells viability when the cells were treated for 4 days (Fig. 4). Cell viability remained close to 100% when the dermal papilla cells were treated with 200 µM testosterone and 250 µg/mL AE extract in combination. VB 62.50 µg/mL was also effective in preventing testosterone induced cell apoptosis, showing results similar to AE extract 250 µg/mL or 75 nM finasteride.    Fig. 5. The study found that both AE extract and VB were safe at concentrations up to 250 µg/mL. Thus, we selected these as the upper limit concentrations for further study on anti-inflammatory activity in macrophage cells.
Effects of AE extract and VB on IL-1β, NO, and TNF-α production in LPS-stimulated macrophages. RAW 264.7 cells were treated by LPS to induce the release of IL-1β, NO and TNF-α, and these cytokines were detected using the ELISA technique. AE ethanolic extract 250 µg/mL or VB 125 µg/mL was selected for IL-1β and NO testing, while both AE extract and VB at 250 µg/mL were used for TNF-α determination. The different concentrations used in this study were taken from the potency screening previously undertaken in our previous work (data not shown). Hydrocortisone was a positive control. The amount of cytokines released from macrophage cells is shown in Figs. 6, 7 and 8. LPS activated pro-inflammatory cytokines such as IL-1β, NO and TNF-α were released from RAW 264.7 cells. Those cytokines can be inhibited by steroid agents such as hydrocortisone, a synthetic preparation of the steroid hormone cortisol, which has an anti-inflammatory response [31][32][33] . Like hydrocortisone 5 µg/mL, AE extract 250 µg/mL showed successful downregulation of IL-1β released from the RAW 264.7 cells treated with LPS ( Fig. 6). Similarly, there was also significant inhibition but to a lesser degree when VB 125 µg/mL was used.
RAW 264.7 cells treated with LPS also produced nitric oxide, which was represented by sodium nitrite content. N(G)-monomethyl-l-arginine (L-NMMA) was used as a positive control. As shown in Fig. 7, the results were that LPS effectively induced the synthesis of sodium nitrite in Raw 264.7 cells. L-NMMA inhibited the synthesis    34 . Both AE extract 250 µg/mL and VB 125 µg/mL also showed greater potency of inhibition of nitric oxide secretion than the LPS control cells.
Tumor necrosis factor (TNF-α) released from LPS treated macrophage cells was strongly inhibited by hydrocortisone ( Fig. 8). Similar to hydrocortisone, 250 μg/mL AE extract and 250 μg/mL VB also significantly suppressed the release of TNF-α.

Effects of AE extract and VB on IL-1α and IL-6 production in UVB irradiated dermal papilla cells.
Dermal papilla cells released IL-1α and IL-6 in the supernatant fluid after being irradiated with 20 J/cm 2 UVA and 100 mJ/cm 2 UVB, respectively. IL-6 and IL-1α levels in the supernatant were measured using cytokine immunoassay (ELISA technique). Hydrocortisone 5 µg/mL was a positive control. The amount of IL-1α is shown in Fig. 9, and the amount of IL-6 is shown in Fig. 10. Figure 9 shows that AE extract 250 µg/mL or VB 125 µg/mL significantly decreased the amount of IL-1α released from the irradiated dermal papilla cells. Similar to IL-1α, AE extract 250 µg/mL and VB 125 µg/mL significantly suppressed the release of IL-6 ( Fig. 10).

Discussion
The bioactivity of AE extract and VB on murine macrophage RAW 267.4 and human dermal papilla cells was evaluated. The RAW 264.7 cells are normally used for evaluating anti-inflammatory activity because the macrophage can be stimulated by LPS to produce IL-1β by activating Toll-like receptors 35,36 . Although the release of pro-inflammatory cytokines in dermal papilla cells has been not fully understood, many reports also reveal the determination of those cytokines from dermal papilla cells and they play a crucial role in dermal papilla cell apoptosis and hair follicular damage 1,8,9,37 . AE extracted with 95% ethanol had the highest content of VB of all tested AE extracts. Both AE extracted with 95% ethanol and VB were non-toxic to the dermal papilla and macrophage cells in the concentration range 0.98 to 500 µg/mL. The inhibition activity of 5α-reductase was evaluated www.nature.com/scientificreports/ by using an enzymatic activity assay. The enzyme used was isolated from LNCaP cell that presents the prominent quantity in 5α-reductase 38,39 . AE extracted with 95% ethanol possessed the highest 5α-reductase inhibition activity (38.26 ± 4.90% at 100 µg/mL). Surprisingly, this activity was not found for VB. These results indicate that other components than VB in the AE extract demonstrate 5α-reductase inhibition activity. Although VB did not appear to affect 5α-reductase inhibition, VB 62.50 µg/mL inhibited dermal papilla cell apoptosis induced with 200 µM testosterone. Similar activity was also found with 250 µg/mL AE extracted with 95% ethanol and 75 nM finasteride. This effect could be due to the down-regulation of cAMP, which prevents cell apoptosis induced by testosterone 29 . AE extract and VB did not show any cytotoxic effects on dermal papilla cells, but these compounds did induce dermal papilla cell proliferation. Cell cycle study indicated that both AE extract and VB at the concentration of 500 µg/mL suppressed dermal papilla cell proliferation (G2/M phase). The increasing numbers of cells in the G1 phase were evidenced for 500 µg/mL AE extract and 500 µg/mL VB, which indicated the induction of G1 phase of dermal papilla cell cycle arrest 40,41 . However, AE extract (125 µg/mL and 250 µg/mL) and VB (62.50 µg/mL and 125 µg/mL) promoted dermal papilla cell proliferation. At these concentrations, the cell numbers in the G2/M or S phases were greatly  Those results were consistent with the results of anti-testosterone activity tests that showed that AE extracts (250 µg/mL) and VB (62.50 µg/mL) prevented cell apoptosis induced by testosterone. The human dermal papilla cell apoptosis induced by testosterone have been well documented 44 . The prevention of cell apoptosis via down-regulating cAMP was reported as a mechanism of VB on testosterone activity 29 . The decrease in testosterone metabolism diminished inflammation initiating cell apoptosis 1,9 . IL-1β and IL-1α are pro-inflammatory cytokines that indicate cells are undergoing an inflammatory process 45 . In various cell types, the IL-1 family can be induced to release several inflammatory cytokines, such as IL-6, prostaglandin E2, tumor necrotic factor-alpha (TNF-α), and nitric oxide (NO) 36,46,47 . The release of NO can be detected by measuring nitrate and nitrite levels in cell culture supernatants. NO is a signaling molecule in the hair follicle. Overexpression of NO is involved in the regulation and differentiation of hair follicles resulting in hair loss 5 . RAW 264.7 cells were used to induce IL-1β, TNF-α and NO-releasing by LPS 35,48 . AE extract 250 µg/ mL and VB 125 µg/mL were observed to inhibit the release of IL-1β from RAW 264.7 cells. Similarly, the release of NO was inhibited by AE extract 250 µg/mL and VB 125 µg/mL. This is in agreement with previous reports which indicated that VB inhibited the expression of pro-inflammatory cytokines in immune cells [49][50][51] . The inhibition occurs when VB binds to the two proinflammatory transcriptional factors, activator protein-1 and NF-κB. This binding results in the downregulation of the two transcription factors 51 and TGF-beta expression 52 , which affects the synthesis of IL-1β and TNF-α. In addition, another publication indicated that VB inhibits NO synthesis by suppressing iNOS enzyme activity 53 . This mechanism can be used to explain the decrease of IL-1β and NO released from RAW 264.7 cells.
Dermal papilla cells can release several inflammatory cytokines. IL-1α, an inflammatory cytokine, was measured in irradiated dermal papilla cells. Generally, IL-1α is found at the cell membrane surface and is released when the cell membrane is damaged 54,55 . UVA 20 mJ/cm 2 was used to induce IL-1α expression in dermal papilla cells. Both AE extract and VB effectively suppressed IL-1α release. IL-6 has been recognized as an androgen inducible negative mediator for AGA development 56 . IL-6 is also a proinflammatory mediator, which is upregulated in the hair follicles of AGA patients. There is a report 57 indicating that hair shaft elongation was inhibited by IL-6 treatment. IL-6 treatment is associated with suppressing the proliferation of matrix cells in cultures of human hair follicles and is known to promote the transition of hair follicles from the anagen to catagen phases in the hair cycle of mice which indicates that shortening the anagen phase results in less time for hair growth.
We induced IL-6 expression by UVB irradiation in the dermal papilla. AE extract 250 µg/mL and VB 125 µg / mL strongly decreased the level of IL-6 released from the irradiated dermal papilla cells. This inhibition could be due to the membrane stabilizing property of VB. We hypothesize that VB forms a stable lipid complex with the cell membrane which may prevent cell membrane damage from UV radiation 58 . These findings suggest that antiinflammatory, 5α-reductase inhibitory, and dermal papilla growth promoter effects of AE extract were the rational use of AE extract in reducing alopecia. Although AE extract contains many active compounds, we are interested in VB and have used it as the biomarker because VB demonstrates an outstanding effect associated with the antihair loss pathway. VB can increase dermal papilla cell proliferation and protection of testosterone-induced dermal www.nature.com/scientificreports/ papilla cell apoptosis concomitantly with anti-inflammation activity. These activities are an influence effect on the protection of hair loss and promote hair regrowth. VB in the AE extract is possible one component among others that contribute to this benefit. Further study needs to be done to identify other bioactive compounds.

Conclusion
AE has been used as a traditional medicine in some Asian countries to treat various diseases. AE also has numerous potential therapeutic effects such as being an anti-inflammatory and skin nourishing agent. VB possesses pharmacologically beneficial qualities for human health, including acting as an antioxidant and anti-inflammatory agent. In summary, AE extract and VB manifest positive effects on human dermal papilla and inflammatory conditions that can be used to reduce hair loss and promote hair growth. Our research suggests that AE extract and VB are potential ingredients for future alopecia therapy.

Materials and methods
Chemicals. 1 were cultured and maintained in a follicle dermal papilla cell growth medium. The growth medium contained growth factors of fetal calf serum 0.04 ml/ml, bovine pituitary extract 0.004 ml/ml, basic fibroblast growth factor recombinant human, 1 ng/ml), and insulin (recombinant human, 5 µg/ml). The cells were incubated under cell culture conditions of 95% relative humidity (RH), 5% CO 2 and 37 °C. They were sub-cultured until they grew to about 80% confluence.
Murine macrophage RAW 264.7 cell culture. The RAW 246.7 (ATCC, Virginia, USA), a murine macrophage cell line, was cultured and maintained in DMEM medium (Gibco, Thermo scientific, MA, USA) containing 10% fetal bovine, 10,000 units/ml of penicillin and streptomycin. The cells were incubated under cell culture conditions of 95% RH, 5% CO 2 and 37 °C. They were sub-cultured until about 80% confluence.
MTT assay for cell viability and proliferation testing. Ten  Determination of 5α-reductase inhibition. Anti-androgenic activity via the steroid 5α-reductase (S5αR) inhibition mechanism was evaluated using a label-free enzymatic inhibitory assay, following the protocol developed by Srivilai and colleagues 38,61,62 . Enzymatic activity was measured by analyzing the DHT formation after enzymatic reaction using liquid chromatography-mass spectrometry (LC-MS).
Enzymatic inhibitory assay. AE extracts were dissolved in DMSO and the inhibition activity determination followed the procedure in Srivilai and colleagues 38,61 . Aliquots of these solutions were added to the assay solution.
The enzymatic reaction was composed of test substances, 34.7 µM testosterone, and 1 mM NADPH in Tris buffer pH 7.4. The reaction was started by adding 200 µL of the homogenate enzyme (75 µg total protein). The mixture was incubated at 37 °C for 60 min. The reaction was stopped by adding 300 µL of hydroxylamine (10 mg/mL in 80% (v/v) ethanol) and incubating at 60 °C for 60 min for the derivatization process. The plate was then centrifuged at 1700 g for 10 min and the supernatants were transferred to another plate ready for injection into the LC-MS. C1 and C2 were the two control samples used. Both controls contained the complete reaction mixture as described above but C1 was stopped before enzymatic incubation, whereas, C2 was stopped after 60 min of incubation. The DHT production was measured using LC-MS. The extracted ion chromatogram (EIC) of derivatized DHT (m/z [M + H] + , 306.2428), the area under the curve was used to calculate enzymatic inhibition: The standard steroid 5α-reductase inhibitor, finasteride, was used as a positive control.
LC-MS method for the measurement of DHT. The determination of DHT was done according to Srivilai and colleagues 38,61 . The Agilent 1260 Infinity Series HPLC system with an auto-sampler accommodating either two 108-vial trays or two 96-well plates (Agilent Technologies, Santa Clara, CA, USA) was used. The analytical reversed-phase column was a Phenomenex Luna® C18 (2) (150 mm × 4.6 mm, 5 µm) with a guard column (Phenomenex C18, 4 mm × 3 mm, 5 µm). The HPLC was connected with an Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA), equipped with dual electrospray ionization (ESI) in positive mode and m/z range 100-1200. Nitrogen was the nebulizing gas at 30 psi, and the drying gas (10 L/ min; 350 °C). The mobile phase was 0.1% (v/v) formic acid in purified water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (LC-MS grade, ACI Labscan, Bangkok, Thailand) as solvent B. The gradient program was used as follows: The initial mobile phase was 60% solvent B and 40% solvent A; solvent B was linearly increased up to 80% over 8 min then held constant for 4 min. Each run was followed by a 2-min post-run. The total runtime analysis was therefore 14 min with the column temperature controlled at 35 °C. The flow rate was 0.5 mL/ min and the injection volume was 20 µL. Mass data were analyzed using Agilent Mass Hunter Qualitative Analysis software version B06.00. The data was identified, calculated and presented in term of inhibition activity.
Determination of IL-1β, NO, and TNF-α secretion inhibition in LPS-stimulated macrophages. One hundred thousand RAW 264.7 cells were seeded into each well of a 24-well plate and incubated under cell culture conditions of 95% relative humidity (RH), 5% CO 2 and 37 °C for 24 h. Next, the cells were treated with 1 µg/ml lipopolysaccharide (LPS) plus 250 µg/ml AE extracts or 125 µg/ml VB or 62.50 µg/ ml VB depending on the tested cytokines. Five micrograms per milliliter of hydrocortisone were the positive control. The treated cells were incubated for 24 h and the supernatants were collected for determination of the amount of IL-1β and TNF-α using an ELISA Kit (Invitrogen, Vienna, Austria). The amount of nitric oxide was determined using the chemical reaction of Griess's reagent (Sigma-Aldrich, Steinheim, Germany).
Determination of IL-1α, IL-6 secretion inhibition in UV radiated dermal papilla cells. One hundred thousand dermal papilla cells were seeded into each well of a 24-well plate and incubated under cell culture conditions of 95% relative humidity (RH), 5% CO 2 and 37 °C for 24 h. The cells were irradiated by 100 mJ/ cm 2 of UVB for the IL-6 study or 20 J/cm 2 of UVA for IL-1α study using UV irradiation chamber (Opsytec, Dr. Grӧbel GmbH, Ettlingen, Germany). The irradiated cells were immediately treated with 250 µg/ml AE extract or 125 µg/ml VB solution and incubated for 24 h. The expression of IL-1 α and IL-6 in the cell culture supernatants were assayed by an ELISA kit according to the manufacturer's protocol (BioLegend, San Diego, CA; Abcam, Cambridge CB2 OAX, UK).